| Objective:To build up a simple assay method for determination of the total alkaloids from Zanthoxylum nitidum(Roxb.)DC.by UV,and then find out a process to extract the total alkaloids with aqueous acid-alcohol and separate them with macroporous resin.Methods:UV method is used to determine the total alkaloids from Zanthoxylum nitidum(Roxb.)DC.The extracting process of the total alkaloids is optimized by orthogonal test with extracting efficacy of total alkaloids as indexes.And then the total alkaloids are separated with macroporous resin at one time or gradiently,prefering the optimal conditions,such as resin,absorption,elution,regeneration,and so on.Finally,the products are analysed by TLC and HPLC.Results:UV method determined the total alkaloids:regression equation was Y=0.08366X+0.0131,r=0.9999,with concentration ranging from 0.51 to 10.20 mg · L-1 while 60%alcohol-pH5.0 acetic acid-sudium acetic buffer as solvent at 328 nm,the average recovery was 99.5%.Optimal extracting process:the process was reflux Zanthoxylum nitidum(Roxb.)DC.granule 3 times,1.0 hour,0.5 hour and 0.5 hour respectively,with 6 times,5 times and 4 times 60%alcohol-0.5%hydrochloric acid as solvent.The extracting efficacy of total alkaloids was 94.65%.Optimal one-time separating process:the extract was recycled ethanol until no alcohol flavor,stayed 2 days,and filtered.Precipitate was dissolved 15 to 20 times with 0.5%hydrochloric acid,filtered.Filtered solution was combined and flowed D-101 macroporous resin column with 8BV(column bed volume),the absorptive rate was 94.81%,the absorptive quantity was 29.45 g · Kg-1.After absorbed 6 hours,elued impurity with 1BV pH5 aqueous hydrochloric acid,eluted alkaloids with 5BV 70%ethanol,the elution rate was 98.60%,the content of total alkaloids in product was 25.19%,the retention rate was 92.92%.Optimal gradient separating process:The first stage:60%alcohol-0.5%hydrochloric acid extract was flowed macroporous resin column 2 times,the absorptive rate was 41.99%,23.25%respectively,the absorptive quantity of primary absorption was 25.08 g · Kg-1,the content of total alkaloids in product was 27.32%,21.91%respectively,in which were ingredients with less polarity,and nitidine chloride was the lest.The second stage:the leaked solution of the first stage second absorption was recycled a specified volume of ethanol,and then was flowed macroporous resin column 2 times,the absorptive rate was 59.34%,44.05%respectively,the absorptive quantity of primary absorption was 15.71 g · Kg-1,the content of total alkaloids in product was 25.22%,17.82%respectively,in which were ingredients with medium polarity,and nitidine chloride was medium.The third stage:the leaked solution of the second stage second absorption was recycled ethanol until no alcohol flavor,and then was flowed macroporous resin column,the absorptive rate was 94.86%,the absorptive quantity was 14.56 g · Kg-1,the content of total alkaloids in product was 18.10%,in which were ingredients with more polarity,and nitidine chloride was the most.The retention rate of alkaloids in whole separating process was 98.07%.Conclusions:The assay method we esteblished is simple,rapid and accurate.The extracting process can improve greatly the extracting efficacy of total alkaloids.The one-time separating process can enrich efficienly total alkaloids,increase retention rate,decrease resin cost,but it is trouble to dissolve the precipitate.The gradient separating process not only resolves the thouble,but also separates diffetent polarity ingredients.The whole process is safe,environmentally friendly and efficient,can be used to extract and separate total alkaloids from Zanthoxylum nitidum(Roxb.)DC.preliminarily.Creative points:pretreatment of sample in determination is simple without combination and extraction,increase work efficiency;added acid into solvent,extracting eff icacy can be improved greatly.It is the first time active ingredient has been separated by gradient absorption. |
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